Recombinant Human XEDAR

Using Recombinant Human XEDAR in research provides a controlled, reproducible tool to study the biological functions of the XEDAR receptor, its signaling pathways, and its involvement in disease, particularly in contexts such as ectodermal dysplasia and potential cancer therapies.

Key reasons to use Recombinant Human XEDAR:

• Functional Studies: Recombinant XEDAR allows precise investigation of its role as a receptor for Ectodysplasin-A2 (EDA-A2), enabling studies on ligand-receptor interactions, downstream signaling, and cellular responses in vitro and in vivo.
• Disease Modeling: Mutations in XEDAR are linked to hypohidrotic ectodermal dysplasia (HED), a disorder affecting hair, teeth, and sweat glands. Recombinant XEDAR can be used to model these diseases, screen for therapeutic agents, and study pathogenic mechanisms.
• Drug Discovery and Screening: Recombinant XEDAR is suitable for high-throughput screening of small molecules or antibodies that modulate its activity, aiding in the identification of potential drug candidates for diseases where XEDAR signaling is implicated.
• Structural Biology: Producing XEDAR recombinantly enables structural studies (e.g., X-ray crystallography, cryo-EM), which are essential for understanding receptor conformation, ligand binding, and for rational drug design.
• Assay Development: Recombinant XEDAR can serve as a standard or positive control in immunoassays (ELISA, western blot, immunohistochemistry), ensuring assay specificity and reproducibility.
• Therapeutic Research: XEDAR has been proposed as a target for gene therapy in cancers such as osteosarcoma, making recombinant forms valuable for preclinical evaluation of therapeutic strategies.

Advantages of using recombinant proteins:

• Consistency and Purity: Recombinant proteins offer high batch-to-batch consistency and purity, reducing experimental variability.
• Human Origin: Recombinant human proteins more closely mimic native human biology compared to animal-derived proteins, improving physiological relevance and translational potential.
• Customization: Recombinant technology allows for the production of wild-type or mutant XEDAR, facilitating studies of disease-associated variants or engineered constructs.

In summary, Recombinant Human XEDAR is a versatile reagent for dissecting XEDAR biology, modeling disease, screening therapeutics, and developing robust assays, all with the reliability and specificity required for advanced biomedical research.

You can use recombinant human XEDAR as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately determined, and it is compatible with your assay antibodies and detection system.

Key considerations and best practices:

• Purity and Characterization: The recombinant XEDAR should be highly purified (typically >95% by SDS-PAGE) and free from contaminants that could interfere with antibody binding or assay performance.
• Concentration Determination: The protein concentration must be accurately measured, ideally by absorbance at 280 nm using a known extinction coefficient, or by amino acid analysis.
• Standard Curve Preparation: Prepare a serial dilution of the recombinant XEDAR in the same buffer as your samples to generate a standard curve for quantification.
• Antibody Compatibility: Ensure that your ELISA antibodies recognize the recombinant form of XEDAR you are using. Some antibodies may only detect native or specific isoforms, so validation is essential.
• Bioactivity: If your assay requires detection of functional (bioactive) XEDAR, confirm that the recombinant protein retains relevant epitopes or activity. Some recombinant proteins are not validated for bioactivity, which may affect quantification if your antibodies are conformation-sensitive.
• Matrix Effects: If you are quantifying XEDAR in complex biological samples (e.g., serum, plasma), perform spike-and-recovery and linearity experiments to confirm that the recombinant standard behaves similarly to endogenous XEDAR in your assay matrix.

Additional notes:

• Many commercial ELISA kits for XEDAR use recombinant human XEDAR as the standard, supporting its suitability for this purpose.
• If you are developing a custom ELISA, you must validate the standard curve and assay performance parameters (sensitivity, specificity, precision, recovery) using your recombinant standard.

In summary, recombinant human XEDAR is appropriate as a standard for ELISA quantification if it is well-characterized, pure, and compatible with your assay system. Always validate its performance in your specific assay context.

Recombinant Human XEDAR has been validated for several key applications in published research, primarily in studies of cancer biology and cell signaling.

• Quantitative Real-Time RT-PCR (qRT-PCR): Used to analyze XEDAR expression levels in human breast cancer cell lines and tumor samples, enabling assessment of gene regulation and correlation with disease states.
• Immunoblotting (Western Blot): Applied to detect XEDAR protein expression in various cell lines and tissue samples, confirming protein presence and relative abundance.
• Methylation-Specific PCR (qMSP): Utilized to examine the methylation status of the XEDAR gene promoter, particularly in the context of epigenetic silencing in cancer. This assay helps distinguish cancerous from non-malignant tissues based on XEDAR promoter methylation.
• Cell Viability and Apoptosis Assays: Recombinant EDA-A2 (the ligand for XEDAR) has been used to stimulate cells expressing XEDAR, with subsequent measurement of cell viability and apoptosis. This validates the functional activity of recombinant XEDAR in mediating cell death signaling pathways.
• Receiver Operator Characteristic (ROC) Curve Analysis: Applied to evaluate the diagnostic utility of XEDAR methylation as a biomarker for distinguishing breast cancer from benign tissue, based on quantitative methylation data.

Additional technical applications mentioned in product and protocol literature (not primary research validation):

• ELISA Standard: Recombinant XEDAR is recommended as a standard for quantifying XEDAR in ELISA assays, supporting its use in protein quantification and biomarker studies.
• SDS-PAGE: Used for purity assessment and molecular weight determination of recombinant XEDAR protein.

Summary Table: Validated Applications for Recombinant Human XEDAR

Key scientific applications focus on cancer biomarker discovery, epigenetic regulation, and cell death signaling. Protocol literature supports additional use in protein quantification and characterization assays.

To reconstitute and prepare Recombinant Human XEDAR protein for cell culture experiments, follow these best-practice steps:

1. Equilibrate and Collect Lyophilized Protein

   • Allow the vial and the reconstitution buffer (e.g., sterile PBS) to reach room temperature.
   • Briefly centrifuge or tap the vial to ensure all lyophilized material is at the bottom.

2. Reconstitution

   • Add sterile buffer (commonly PBS or as specified in the product datasheet) to achieve the desired concentration, typically 100 μg/mL is standard for similar TNF receptor family proteins.
   • If the datasheet recommends, include at least 0.1% carrier protein (such as BSA or HSA) in the buffer to stabilize the protein and prevent adsorption to surfaces.
   • Gently pipette or swirl to dissolve. Do not vortex or shake vigorously, as this can denature the protein.

3. Incubation

   • Let the solution sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.

4. Aliquot and Storage

   • Use the reconstituted protein immediately for experiments, or aliquot into single-use portions to avoid repeated freeze-thaw cycles.
   • For short-term storage (up to 1 week), keep at 2–8°C.
   • For long-term storage, freeze aliquots at –20°C to –80°C. Addition of carrier protein (e.g., 0.1% BSA) and/or glycerol (5–50%) is recommended for stability.
   • Avoid repeated freeze-thaw cycles, as this can degrade the protein.

5. Preparation for Cell Culture

   • Before adding to cell culture, dilute the reconstituted stock to the working concentration using cell culture medium (with or without serum, as appropriate for your assay).
   • If using serum-free conditions, avoid animal-derived carriers and consider using trehalose as a stabilizer.

Key technical notes:

• Always consult the specific Certificate of Analysis (CoA) or datasheet for your XEDAR protein lot for any unique instructions.
• Use sterile technique throughout to prevent contamination.
• If the protein is to be used in sensitive or animal-free systems, avoid BSA/FBS and use non-animal stabilizers.

Summary Table: Recombinant Human XEDAR Reconstitution

These steps ensure maximum stability and bioactivity of recombinant XEDAR for cell culture applications.